Project Summary: A phenotypic screen for osteoarthritic pain therapeutics using all- optical electrophysiology Chronic pain affects over 100 million adults in the United States, many of whom cannot find relief with current medications. Of these patients, nearly 1/3 of them suffer from osteoarthritis. Current treatments for chronic pain include opioids and non-steroidal inflammatory agents. However, efficacy of these drugs in chronic treatment is restricted by the development of tolerance and dose-limiting toxicities. Opioids in particular are highly addictive, and dependency has caused a large societal burden. Despite the clear, unmet medical need and significant research activity, few new classes of non-opioid drugs targeting chronic pain have appeared in the past decade. Quell Therapeutics uses the Optopatch platform for making all-optical electrophysiology measurements in neurons at a throughput sufficient for phenotypic screening. Using engineered optogenetic proteins, blue and red light can be used to stimulate and record neuronal activity, respectively. Custom microscopes enable electrophysiology recordings from 100?s of individual neurons in parallel with high sensitivity and temporal resolution, a capability currently not available with any other platform screening technology. Here, we combine the Optopatch platform with an in vitro model of chronic pain, where dorsal root ganglion (DRG) sensory neurons are bathed in a mixture of inflammatory mediators found in the joints of osteoarthritis patients. The neurons treated with the inflammatory mixture become hyperexcitable, mimicking the anticipated cellular pain response. We calculate the functional phenotype of arthritis pain, which captures the difference in action potential shape and firing rate in response to diverse stimuli. We will screen for small molecule compounds that reverse the pain phenotype while minimizing perturbation of neuronal behavior orthogonal to the pain phenotype, the in vitro ?side effects.? We will counter-screen against neurons from the cerebral cortex and cardiomyocytes from the heart to prioritize compounds that act selectively in inflamed sensory neurons. The highest ranking compounds will be chemically optimized and their pharmacokinetic, drug metabolism, and in vivo efficacy will be characterized. Our goal is to advance therapeutic discovery for pain, which may ultimately help relieve the US opioid crisis.